Synchronization of ion generation with cycling of a discontinuous atmospheric interface

ABSTRACT

The invention generally relates to methods and devices for synchronization of ion generation with cycling of a discontinuous atmospheric interface. In certain embodiments, the invention provides a system for analyzing a sample that includes a mass spectrometry probe that generates sample ions, a discontinuous atmospheric interface, and a mass analyzer, in which the system is configured such that ion formation is synchronized with cycling of the discontinuous atmospheric interface.

RELATED APPLICATION

The present application is a continuation of U.S. nonprovisionalapplication Ser. No. 14/052,815, filed Oct. 14, 2013, which is acontinuation-in-part of U.S. nonprovisional application Ser. No.13/887,911, filed May 6, 2013, which is a continuation-in-part ofPCT/US12/21964, filed Jan. 20, 2012, which claims the benefit of andpriority to U.S. provisional patent application Ser. No. 61/434,473,filed Jan. 20, 2011, the content of each of which is incorporated byreference herein in its entirety.

GOVERNMENT SUPPORT

This invention was made with U.S. government support underN00014-05-1-0454 awarded by U.S. Office of Naval Research and CHE0848650awarded by National Science Foundation. The U.S. government has certainrights in the invention.

FIELD OF THE INVENTION

The invention generally relates to methods and devices forsynchronization of ion generation with cycling of a discontinuousatmospheric interface.

BACKGROUND

Mass spectrometry (MS) plays an important role in chemical analysiswhich is currently being enhanced by the increasing demand for rapidtrace analysis in the areas of public safety, forensics, food safety andpharmaceutical quality assurance, amongst others. These demands haveproduced a need to simplify MS instrumentation and methodologies. Thisin turn has resulted in the development of miniaturized instrumentation(Gao et al., Anal. Chem. 2006, 78, 5994-6002; Cotte-Rodriguez et al.,Analyst 2006, 131, 579-589; and Cotte-Rodriguez et al., Anal. Chem.2008, 80, 1512-1519) and development of ambient ionization methods inwhich samples are examined without preparation in their native state(Venter et al., TrAC, Trends Anal. Chem. 2008, 27, 284-290; Cooks etal., Biopolymers 2009, 92, 297-297; Ifa et al., Analyst 2010, 135,669-681; Shiea et al., Rapid Commun. Mass Spectrom. 2005, 19, 3701-3704;Huang et al., Annu. Rev. Anal. Chem. 2010, 3, 43-65; Chen et al., J AmSoc Mass Spectrom 2009, 20, 1947-1963; Law et al., Angew. Chem., Int.Ed. 2009, 48, 8277-8280; Chingin et al., Angew. Chem., Int. Ed. 2010,49, 2358-2361; and Weston, Analyst 2010, 135, 661-668). Particularly,miniaturized instrumentation is being combined with ambient ionizationmethods to produce mass spectrometers that can be easily used outside oflaboratories.

However, a problem with a system that combines miniaturizedinstrumentation and ambient ionization is that such a system is limitedby the low pumping speed of small mass spectrometers and the largenebulizing gas and solvent volumes that must be handled. This problemhas been addressed by the development of a discontinuous atmosphericpressure interface (DAPI; Gao et al., Anal. Chem. 2008, 80, 4026-4032;Gao et al., Int. J. Mass Spectrom. 2009, 283, 30-34; and Gao et al.,Int. J. Mass Spectrom. 2009, 283, 30-34). The DAPI interface is openedbriefly to admit a bolus of ions, solvent vapor and gas, then closedwhile the neutrals are pumped away before the trapped ions are massanalyzed.

Even with the implementation of a DAPI, there is still a need toincrease the sensitivity and sampling efficiency of systems that combineminiaturized instrumentation and ambient ionization.

SUMMARY

The invention recognizes that synchronizing ion generation with cyclingof a discontinuous atmospheric interface provides a system with improvedsensitivity, reduced solvent usage, reduced nebulizing gas usage, andimproved sampling efficiency compared to previous systems in which iongeneration is continuous and operates independently of cycling of thediscontinuous atmospheric pressure interface. In this manner, systems ofthe invention provide a more sensitive and more efficient massspectrometer. Particularly, systems of the invention are well suited foruse outside of laboratories and at the location of the sample, e.g., acrime scene, a food processing facility, or a security check-point at anairport.

In certain aspects, the invention provides a system for analyzing asample that includes a mass spectrometry probe that generates sampleions, a discontinuous atmospheric interface, and a mass analyzer, inwhich the system is configured such that ion formation is synchronizedwith cycling of the discontinuous atmospheric interface. In certainembodiments, the probe includes a spray emitter and a high voltagesource, in which the probe is configured such that the high voltagesource is not in contact with spray emitted by the spray emitter. Incertain embodiments, the ions are generated by inductive charging. Suchan inductive charging probe is shown herein to be interfaced with adiscontinuous atmospheric pressure interface, however, such products canbe directed interfaced with any type of mass spectrometer without theuse of a discontinuous atmospheric pressure interface.

In other aspects, the invention provides a method for analyzing a samplethat involves generating ions of an analyte in a sample using a massspectrometry probe, discontinuously directing the ions into a massanalyzer, and analyzing the ions, in which the generating step issynchronized with the directing of the ions into the mass analyzer.Discontinuous atmospheric pressure interfaces and methods fordiscontinuously directing ions into a mass analyzer are described inU.S. Pat. No. 8,304,718, the content of which is incorporated byreference herein in its entirety.

The mass spectrometry probe may be any probe known in the art. Incertain embodiments, the probe operates by a direct ambient ionizationtechnique. Exemplary mass spectrometry techniques that utilize directambient ionization/sampling methods including desorption electrosprayionization (DESI; Takats et al., Science, 306:471-473, 2004 and U.S.Pat. No. 7,335,897); direct analysis in real time (DART; Cody et al.,Anal. Chem., 77:2297-2302, 2005); Atmospheric Pressure DielectricBarrier Discharge Ionization (DBDI; Kogelschatz, Plasma Chemistry andPlasma Processing, 23:1-46, 2003, and PCT international publicationnumber WO 2009/102766), and electrospray-assisted laserdesoption/ionization (ELDI; Shiea et al., J. Rapid Communications inMass Spectrometry, 19:3701-3704, 2005). The content of each of thesereferences in incorporated by reference herein its entirety. Inparticular embodiments, the direct ambient ionization technique isdesorption electrospray ionization.

In other embodiments, the probe operates by electrospray ionization. Inother embodiments, the probe is a paper spray probe (internationalpatent application number PCT/US10/32881). In other embodiments, theprobe is a low temperature plasma probe. Such probes are described inU.S. patent application Ser. No. 12/863,801, the content of which isincorporated by reference herein in its entirety.

In other embodiments, the system further includes a source of nebulizinggas. In certain embodiments, the source of nebulizing gas is configuredto provide pulses of gas. Generally, the gas pulses are alsosynchronized with ion formation and cycling of the discontinuousatmospheric interface.

In certain embodiments, discontinuously directing ions into the massanalyzer may involve opening a valve connected to an atmosphericpressure interface, wherein opening of the valve allows for transfer ofions substantially at atmospheric pressure to the mass analyzer atreduced pressure, and closing the valve connected to the atmosphericpressure interface, wherein closing the valve prevents additionaltransfer of the ions substantially at atmospheric pressure to the massanalyzer at reduced pressure.

The mass analyzer may be for a mass spectrometer or a miniature orhandheld mass spectrometer. Exemplary mass analyzers include aquadrupole ion trap, a rectalinear ion trap, a cylindrical ion trap, aion cyclotron resonance trap, or an orbitrap. An exemplary miniaturemass spectrometer is a handheld rectilinear ion trap mass spectrometer,which is described, for example in Gao et al. (Anal. Chem.,80:7198-7205, 2008), Hou et al. (Anal. Chem., 83:1857-1861, 2011), andSokol et al. (Int. J. Mass Spectrom., In Press, Corrected Proof, 2011),the content of each of which is incorporated herein by reference hereinin its entirety.

Another aspect of the invention provides a method for forming sampleions that involves flowing a sample through a device, pulsing voltagefrom a source that is not in contact with the flowing sample toinductively interact with the flowing sample, thereby producing sampleions. In certain embodiments, the device is a probe that operates by adirect ambient ionization technique, such as desorption electrosprayionization. In other embodiments, the probe operates by electrosprayionization. In other embodiments, the probe is a paper spray probe. Inother embodiments, the probe is a low temperature plasma probe. Theprobe may be interfaced with a discontinuous atmospheric pressureinterface or directly interfaced with an ion analyzing device, such as amass spectrometer.

Another aspect of the invention provides a method for synchronizingsample ion generation from a mass spectrometry probe with cycling of adiscontinuous atmospheric interface, involving generating a sample sprayfrom a mass spectrometry probe, pulsing voltage from a source that isnot in contact with the sample spray to inductively interact with thesample spray, thereby producing sample ions, and synchronizing thepulsing of the voltage with the cycling of a discontinuous atmosphericinterface. Methods of the invention may further involve pulsingnebulizing gas to interact with the sample, in which the gas pulses arealso synchronized with ion formation and cycling of the discontinuousatmospheric interface.

Another aspect of invention provides a method for applying high voltageon electrospray/nanoelectrospray/paper spray tips without physicalcontact. The induced high voltage leads to burst of droplets inelectrospray/nanoelectrospray/paper spray (international patentapplication number PCT/US10/32881), and the frequency of the spray isthat of the applied potential. Methods of the invention may also be usedwith low temperature plasma probes. Such probes are described in U.S.patent application Ser. No. 12/863,801, the content of which isincorporated by reference herein in its entirety.

Another aspect of the invention provides a method for producing bothpositive and negative ions in a sample spray that involves applying apulsed voltage to a sample spray from an electrode that is not incontact with the spray to produce both positive and negative ions in thespray. The method may further involve recording mass spectra of thepositive and negative ions. Recording may involve switching polarity ofa mass spectrometer while the mass spectrometer is receiving the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a schematic diagram of system and method of the inventionusing a miniature MS in which charged droplet creation, nebulizing gaspulsing and sample introduction into the MS are all synchronized. FIG.1B shows a pulse sequence used in synchronized experiment. FIGS. 1C-1Dshow an average of 5 DESI mass spectra recorded for cocaine on a glasssubstrate, spray solvent MeOH/water (0.5 μL/min) using a mini MSinterfaced to a DAPI operated at a duty cycle of 1:100. FIG. 1C showssynchronized DESI/DAPI-Mini experiment using 10 ng cocaine. FIG. 1Dshows a conventional experiment using 1,000 ng cocaine.

FIG. 2A shows measurement of nano sprayer voltage and current. FIG. 2Bshows induced voltage recorded inside the DESI source when a nearbyelectrode voltage is pulsed. FIG. 2C shows synchronized DESI massspectra of MRFA (MET-ARG-PHE-ALA) (20 ng on glass) showing bothpolarities recorded in successive scans made at 5 Hz without changingion source potentials.

FIGS. 3A-C show nanoelectrospray using 1 μg/mL methamphetamine inMeOH/water with DAPI interface (duty cycle 1:100) on a Mini 10,averaging signal for 5 min. FIG. 3A shows synchronized experiment (80 pLper scan, flow rate 5 nL/min). FIG. 3B shows conventional experiment(6.5 nL, flow rate 400 nL/min). FIG. 3C shows synchronized electrosprayMS of 100 ng/mL Ultra mark 1621 recording both polarities in successivescans without any changes in ion source potentials using bench top MS.

FIG. 4 shows voltage curves measured inside DESI spray emitter tip.Left: single conventional contact DC pulse (3 kV) applied to thesolution for 11 msec (rise time ˜20 ms, fall time ca. 250 ms). Middle:pulsed inductive DC (5 kV) applied for 11 ms at 5,000 Hz. Right: pulsedinductive DC (5 kV) applied for 11 ms at 1000 Hz. Induced potentialswere applied to the outer metal capillary of the DESI source and riseand fall time were less than 1 ms.

FIG. 5, Top: Synchronized DESI/Mini MS spectrum of 1 ng methamphetamine(m/z 150); Bottom: conventional DESI/Mini MS spectrum of 100 ngmethamphetamine (m/z 150).

FIG. 6 shows a chronogram of selected ion (m/z 304 for cocaine) usingsolid curve): 1,000 ng sample which lasts for 15 seconds usingconventional DESI/DAPI/Mini 10 and dashed curve): 10 ng sample whichlasts for 25 seconds using synchronized DESI/DAPI/Mini 10.

FIGS. 7A-B show total ion chronogram for conventional (contact DC) nanoESI of solution containing both cocaine (300 ng/mL) andp-toluenesulfonic acid (100 ng/mL). FIG. 7A shows an MS detectoroperating in positive mode with 5 positive pulses high voltage (1.4 kV)pulses applied to conventional nano ESI emitter (solution contact)followed by 9 negative pulses (−1.4 kV), insert: protonated ion ofcocaine (m/z 304). FIG. 7B shows an MS detector operating in negativemode with 5 positive pulses high voltage (1.4 kV) applied toconventional nano ESI emitter (solution contact) followed by 9 negativepulses (−1.4 kV), insert: deprotonated ion of p-toluenesulfonic acid(m/z 171).

FIG. 8 shows synchronized ESI detection using bench top MS for solutioncontaining p-toluene sulfonic acid (20 ng/mL), propranolol (50 ng/mL)and atenolol (70 ng/mL). Above: spectrum recorded when MS detectoroperating in negative mode and deprotonated p-toluene sulfonic acid (m/z171) was detected. Below: spectrum recorded when MS detector operatingin positive mode and protonated propranolol (m/z 260) and atenolol (m/z267) were detected.

FIG. 9 shows comparison of conventional and synchronized nano ESI MSspectrum of 2 μg/mL reserpine. Top: Conventional nano ESI withoxidization product detected (m/z 625), similar to the results fromPeintler-Krivan et al. (Rapid Communications in Mass Spectrometry 2010,24, 1327-1334). Bottom: Synchronized inductive nano ESI MS spectrum.

FIG. 10 shows a schematic showing a discontinuous atmospheric pressureinterface coupled in a miniature mass spectrometer with rectilinear iontrap.

FIG. 11 shows a schematic showing a spray device for generating anddirecting a DESI-active spray onto sample material (analyte) and forcollecting and analyzing the resulting desorbed ions.

FIG. 12 shows a schematic showing an embodiment of a low temperatureplasma (LTP) probe.

FIG. 13A shows a schematic of a sample solution being fed to a piece ofpaper for electrospray ionization. FIG. 13B shows a schematic of asample solution pre-spotted onto the paper and a droplet of solventbeing subsequently supplied to the paper for electrospray ionization.

FIG. 14A shows a schematic of high throughput inductive nESI ion sourcearray (rotating form). FIG. 14B shows inductive nESI ion source array(linear form), Insert: image of the inductive nESI plume. Appliedvoltage pulse train (10-3000 Hz, 2-4 kV).

FIG. 15 is a schematic showing an inductive ESI-based on-line reactionmonitoring system.

FIG. 16 panels A-F show time-resolved mass spectra of reductiveamination of 4-pyridine-carboxaldehyde (2) with 4-(aminomethyl)pyridine(1). Reagent 1 was injected into the reaction solvent at 3 min.

FIG. 17 is a selective ion chronogram showing protonated 1disappearance, formation and disappearance of 3, and formation of 4after NaBH₄ introduction.

FIG. 18 is a set of graphs showing kinetic curves of compounds 1, 3, 4in reductive amination of 4-pyridine-carboxaldehyde with4-(aminomethyl)pyridine monitored by ¹H NMR.

FIG. 19 shows a selected ion chronogram of 4-(aminomethyl)pyridine (1)when measuring the delay time of inductive ESI-MS monitoring system.

FIG. 20 panels A-H show time-resolved mass spectra of Pd—C catalyzedhydrogenolysis of benzaldehydes reaction mixture. Precursor 5 wasinjected into the suspension of Pd/C in methanol at 2 min.

FIGS. 21A-B show a selective ion chronogram of the precursor 5,intermediate 7, 8, 9 and product 6 in the reaction of Pd—C catalyzedhydrogenolysis of 3,4-dimethoxy-benzaldehyde. FIG. 21A is a time rangeof 0-130 min. FIG. 21B is a time range of 2.5-3.5 min.

FIG. 22 is a total ion chronogram and selective ion chronograms showing3-bromoquinoline (m/z 208), the product of Pd-PEPPSI (m/z 389), arepresentative intermediate (m/z 407) and the reaction productethylquinoline (m/z 158) in the Negishi cross coupling of3-bromoquinoline with diethyl zinc catalyzed by Pd-PEPPSI in THF usingthe inductive ESI apparatus described herein.

FIG. 23 is an inductive ESI mass spectrum of the Negishi cross couplingreaction mixture of 3-bromoquinoline with diethylzinc catalyzed byPd-PEPPSI in THF recorded at 12.92 min.

DETAILED DESCRIPTION

The invention generally relates to methods and devices forsynchronization of ion generation with cycling of a discontinuousatmospheric interface. In certain embodiments, the invention provides asystem for analyzing a sample that includes a mass spectrometry probethat generates sample ions, a discontinuous atmospheric interface, and amass analyzer, in which the system is configured such that ion formationis synchronized with cycling of the discontinuous atmospheric interface.An exemplary system is shown in FIG. 1. The system includes a massspectrometry probe that operates by an ambient ionization method.Ambient ionization methods include spray-based (Cooks et al., Science2006, 311, 1566-1570; Takats et al., Science 2004, 306, 471-473; Talatyet al., Analyst 2005, 130, 1624-1633; Liu et al., Anal. Chem. 2010, 82,2463-2471; Wang et al., Angew. Chem., Int. Ed. 2010, 49, 877-880;Kertesz et al., Anal. Chem. 2008, 80, 1027-1032; Kertesz et al., Anal.Chem. 2008, 80, 5168-5177; and Bereman et al., J. Am. Soc. MassSpectrom. 2007, 18, 1093-1096) plasma-based (Cody et al., Anal. Chem.2005, 77, 2297-2302; and Block et al., J. Agric. Food Chem. 2010, 58,4617-4625) and laser-assisted methods (Brady et al., Rapid Commun. MassSpectrom. 2010, 24, 1659-1664; Judge et al., Anal. Chem. 2010, 82,3231-3238; Nemes et al., Anal. Chem. 2007, 79, 8098-8106; Nemes et al.,Anal. Chem. 2008, 80, 4575-4582; and Nemes et al., Anal. Chem. 2009, 81,6668-6675).

Like other ambient methods, desorption electrospray ionization (DESI)has the advantages of simple instrumentation, rapid and sensitiveanalysis, and broad applicability. Synchronized inductive DESI showsgood performance: i) over 100-fold improvement in sensitivity (FIGS. 1cand 1d ) while still using the 1:100 DAPI duty cycle, ii) reducedsolvent spray flow rate from ˜5 μL/min to ˜0.5 μL/min, iii) reducednebulizing gas usage from ca. 2 to 0.2 L/min, iv) improved samplingefficiency by a factor of 100 and v) quasi-simultaneous recording ofpositive and negative ion spectra using a pulsed monopolar ion source.

FIG. 1 shows a system set-up in which a DESI probe includes a sprayemitter and a high voltage source, in which the probe is configured suchthat the high voltage source is not in contact with spray emitted by thespray emitter. In this manner, the ions are generated by inductivecharging, i.e., an inductive method is used to charge the primarymicrodroplets. This allows droplet creation to be synchronized with theopening of the sample introduction system (and also with the pulsing ofthe nebulizing gas). The generated ions are directed into the adiscontinuous atmospheric interface, a distal end of which is operablycoupled to a mass spectrometer. In other embodiments, the inductiveprobe is directly interfaced with a mass spectrometer and used without adiscontinuous atmospheric interface.

Synchronization of ion generation with the cycling of the DAPI is basedon accurate control of charged droplet creation by placing an electrodenear a spray emitter (typically 2-5 mm distant) and pulsing itrepetitively to high positive potentials (5-7 kV, 50-3,000 Hz, pulsewidth ˜0.2-2 ms). The pulsed positive voltage was applied to a metaltube (id 250 μm), covering an inner silica capillary which served as thespray emitter tip (id 50 μm). Electromagnetic induction produces highelectrical fields in the DESI source that result in bursts of chargeddroplets. Precise synchronization with the DAPI interface is possiblebecause the inductive pulsed DC high voltage has the necessary shorton/off response times of ca. 1 millisecond (timing control datacomparing inductive and conventional contact DC sprays are shown in FIG.2B and FIG. 4). The nebulizing gas flow was also synchronized to the MSscan function (FIG. 1B). The DAPI pinch valve was opened for the first10 ms while ions were being admitted into the MS then closed for theremainder of the scan period. Both the spray voltage and nebulizing gaswere triggered on 20 ms before the pinch valve was opened, and remainedopen for the 10 ms during the ion introduction period. The spraysolution flow rate was set at 0.5 μL/min. Other DESI conditions in thesynchronized experiment remained the same as in the conventional DESIexperiment (see Example 1 and Table 1 below).

TABLE 1 Typical synchronized and conventional DESI source settingsSynchronized DESI conventional DESI Spray voltage 3.5-6 kV ^(a) 4-5 kVSolvent flow rate 0.1-0.5 μL/min 2-5 μL/min Nebulizating gas flow 0.15L/min ^(b) 2.1 L/min rate Spray polarity Provide both positiveswitchable and negative ions Angle between source 40° 40° and sampleDistance between source 2 mm 2 mm and sample Distance between 3 mm 3 mmsample and MS inlet DAPI opening time 11 ms 11 ms Ion production duty 3%100% cycle Ion injection duty cycle 1%  1% ^(a) Inductive voltagemeasured inside DESI emitter (peak-peak) ^(b) Average gas flow rate

FIGS. 1C and 1D provide data showing the improved sensitivity andsampling efficiency of systems of the invention compared to conventionalambient ionization systems. FIG. 1C shows that 1 μg of cocaine is neededto record a DESI spectrum in the conventional continuous mode comparableto that given by 10 ng cocaine in the synchronized mode (FIG. 1D). Thisand similar results for other compounds (atenolol, methamphetamine andmorphine) indicate an approximately two orders of magnitude increase insensitivity for synchronized DESI over conventional DESI using aminiature MS (FIG. 5).

In addition to the decreased detection limits, synchronized DESI alsoprovides higher sampling efficiency. For conventional DESI, 1 μg cocainesignal lasted for ca. 15 seconds, while with synchronization just 10 ngof sample provides signal for the same period (FIG. 6). The improvementof two orders of magnitude in sensitivity is particularly important forsamples of small size, where ionization efficiency is most important.Other improvements due to synchronization include the decreasednebulizing gas flow rate from ˜2.1 L/min to ˜0.15 L/min and the spraysolution flow rate decrease from 5 μL/min to 0.5 μL/min.

As important as is the improved analytical performance, is the newcapabilities achieved in terms of virtually simultaneous production ofions of both positive and negative polarity from a single spray emitterwithout changing the polarity of the applied potential. This capabilityis illustrated by the spectrum obtained for the tetrapeptide MRFA (FIG.2C). The protonated molecule appears in the positive mode and thedeprotonated form when the polarity of the detector is switched tonegative. Detector switching can be done at 1 Hz, fast enough to recordspectra of alternating polarities in successive scans. By contrast,conventional pulsed DC electrospray (Maheshwari et al., Appl. Phys.Lett. 2006, 89. 234103; and Chetwani et al., J. Am. Soc. Mass Spectrom.2010, 21, 1852-1856) provided ions with either positive or negative, butnot both polarities (FIG. 7). This bipolar capability is based on thecharacteristics of the voltages involved in inductive DESI. The inducedpotential measured inside the DESI spray emitter during the synchronizedexperiment was found to have the same frequency as the pulsed voltageapplied to the outer electrode of the source and an amplitude of 1.2-2kV, similar to that used in the normal contact experiments (FIGS. 2A-B).However the induced voltage inside the emitter shows ringing with bothpositive and negative components and a peak-to-peak voltage of ca. 3 kV.The short pulse width of the repetitively pulsed (5-2,000 Hz) positivepotential applied to the outer electrode caused the induced potential toswing from high positive to high negative values in 1 ms. An apparentlystable electrospray plume could be observed, indicating that the inducedpotential is high enough to generate an electrospray, similar to thatachieved in a direct contact AC electrospray experiment (Maheshwari etal., Appl. Phys. Lett. 2006, 89. 234103; and Chetwani et al., J. Am.Soc. Mass Spectrom. 2010, 21, 1852-1856). The result is that bothpositive and negative ions can be observed simply by switching thepolarity of the mass spectrometer, without making any ion source changesby rapidly polarizing the spray solution in opposite polarities. Thesenew capabilities should facilitate rapid chemical identification andminimize prior sample manipulation.

FIG. 3 compares the performance of conventional and synchronizednanoelectrospray coupled to a Mini 10. Similar ion intensities and S/Nratios are achieved even though synchronized spray rates are ˜80 timeslower, corresponding to an 80-fold improvement in sampling efficiency.This highlights an advantage of synchronized ESI or DESI in applicationswhen the sample amount is limited, as in single cell mass spectrometry.The ability to detect ions of both polarities extends to electrosprayionization. Using Ultra mark 1621 (FIG. 3C) as an example, both positiveand negative ions can be detected when the synchronized ESI experimentis performed on a commercial benchtop instrument. Similar results wereobserved for p-toluenesulfonic acid, propranolol and atenolol (FIG. 8).Another advantage of the fast switching of the polarity of the inducedpotential inside the spray emitter was the elimination of unwantedelectrochemical reactions during DESI/ESI (FIG. 9).

In summary, both DESI and ESI benefit in terms of improved sensitivityfrom controlled droplet generation which is available through the use ofinduced rather than directly applied potentials (Tu et al., J. Am. Soc.Mass Spectrom. 2008, 19, 1086-1090). These advantages also extend toother ambient ionization methods including plasma-based methods.Synchronization of droplet creation with ion transfer into a miniaturemass spectrometer reduces nebulizing gas and solution flow rates by anorder of magnitude, and improves in-situ operation. Synchronized DESIalso offers significant new capabilities in temporal control of ionpolarity on a scan-to-scan basis with millisecond inversion of solutionpolarity. Recent interest in DESI measurements on the millisecond timescale (Barbula et al., Anal. Chem. 2009, 81, 9035-9040) and in the studyof intermediates in solution-phase reactions while sampling on themillisecond time (Perry et al., Angew. Chem., Int. Ed. 2010, in press)might benefit from the bipolarity and enhanced sensitivity of thepresent methodology.

Discontinuous Atmospheric Pressure Interface (DAPI)

Discontinuous atmospheric interfaces are described in Ouyang et al.(U.S. Pat. No. 8,304,718 and PCT application number PCT/US2008/065245),the content of each of which is incorporated by reference herein in itsentirety.

An exemplary DAPI is shown in FIG. 10. The concept of the DAPI is toopen its channel during ion introduction and then close it forsubsequent mass analysis during each scan. An ion transfer channel witha much bigger flow conductance can be allowed for a DAPI than for atraditional continuous API. The pressure inside the manifold temporarilyincreases significantly when the channel is opened for maximum ionintroduction. All high voltages can be shut off and only low voltage RFis on for trapping of the ions during this period. After the ionintroduction, the channel is closed and the pressure can decrease over aperiod of time to reach the optimal pressure for further ionmanipulation or mass analysis when the high voltages can be is turned onand the RF can be scanned to high voltage for mass analysis.

A DAPI opens and shuts down the airflow in a controlled fashion. Thepressure inside the vacuum manifold increases when the API opens anddecreases when it closes. The combination of a DAPI with a trappingdevice, which can be a mass analyzer or an intermediate stage storagedevice, allows maximum introduction of an ion package into a system witha given pumping capacity.

Much larger openings can be used for the pressure constrainingcomponents in the API in the new discontinuous introduction mode. Duringthe short period when the API is opened, the ion trapping device isoperated in the trapping mode with a low RF voltage to store theincoming ions; at the same time the high voltages on other components,such as conversion dynode or electron multiplier, are shut off to avoiddamage to those device and electronics at the higher pressures. The APIcan then be closed to allow the pressure inside the manifold to dropback to the optimum value for mass analysis, at which time the ions aremass analyzed in the trap or transferred to another mass analyzer withinthe vacuum system for mass analysis. This two-pressure mode of operationenabled by operation of the API in a discontinuous fashion maximizes ionintroduction as well as optimizing conditions for the mass analysis witha given pumping capacity.

The design goal is to have largest opening while keeping the optimumvacuum pressure for the mass analyzer, which is between 10-3 to 10-10torr depending the type of mass analyzer. The larger the opening in anatmospheric pressure interface, the higher is the ion current deliveredinto the vacuum system and hence to the mass analyzer.

An exemplary embodiment of a DAPI is described herein. The DAPI includesa pinch valve that is used to open and shut off a pathway in a siliconetube connecting regions at atmospheric pressure and in vacuum. Anormally-closed pinch valve (390NC24330, ASCO Valve Inc., Florham Park,N.J.) is used to control the opening of the vacuum manifold toatmospheric pressure region. Two stainless steel capillaries areconnected to the piece of silicone plastic tubing, the open/closedstatus of which is controlled by the pinch valve. The stainless steelcapillary connecting to the atmosphere is the flow restricting element,and has an ID of 250 μm, an OD of 1.6 mm ( 1/16″) and a length of 10 cm.The stainless steel capillary on the vacuum side has an ID of 1.0 mm, anOD of 1.6 mm ( 1/16″) and a length of 5.0 cm. The plastic tubing has anID of 1/16″, an OD of ⅛″ and a length of 5.0 cm. Both stainless steelcapillaries are grounded. The pumping system of the mini 10 consists ofa two-stage diaphragm pump 1091-N84.0-8.99 (KNF Neuberger Inc., Trenton,N.J.) with pumping speed of 5 L/min (0.3 m3/hr) and a TPD011 hybridturbomolecular pump (Pfeiffer Vacuum Inc., Nashua, N.H.) with a pumpingspeed of 11 L/s.

When the pinch valve is constantly energized and the plastic tubing isconstantly open, the flow conductance is so high that the pressure invacuum manifold is above 30 torr with the diaphragm pump operating. Theion transfer efficiency was measured to be 0.2%, which is comparable toa lab-scale mass spectrometer with a continuous API. However, underthese conditions the TPD 011 turbomolecular pump cannot be turned on.When the pinch valve is de-energized, the plastic tubing is squeezedclosed and the turbo pump can then be turned on to pump the manifold toits ultimate pressure in the range of 1×10 5 torr.

The sequence of operations for performing mass analysis using ion trapsusually includes, but is not limited to, ion introduction, ion coolingand RF scanning. After the manifold pressure is pumped down initially, ascan function is implemented to switch between open and closed modes forion introduction and mass analysis. During the ionization time, a 24 VDC is used to energize the pinch valve and the API is open. Thepotential on the rectilinear ion trap (RIT) end electrode is also set toground during this period. A minimum response time for the pinch valveis found to be 10 ms and an ionization time between 15 ms and 30 ms isused for the characterization of the discontinuous API. A cooling timebetween 250 ms to 500 ms is implemented after the API is closed to allowthe pressure to decrease and the ions to cool down via collisions withbackground air molecules. The high voltage on the electron multiplier isthen turned on and the RF voltage is scanned for mass analysis. Duringthe operation of the discontinuous API, the pressure change in themanifold can be monitored using the micro pirani vacuum gauge (MKS 925C,MKS Instruments, Inc. Wilmington, Mass.) on Mini 10.

Desorption Electrospray Ionization

Desorption electrospray ionization (DESI) is described for example inTakats et al. (U.S. Pat. No. 7,335,897), the content of which isincorporated by reference herein in its entirety. DESI allows ionizingand desorbing a material (analyte) at atmospheric or reduced pressureunder ambient conditions. A DESI system generally includes a device forgenerating a DESI-active spray by delivering droplets of a liquid into anebulizing gas. The system also includes a means for directing theDESI-active spray onto a surface. It is understood that the DESI-activespray may, at the point of contact with the surface, include both oreither charged and uncharged liquid droplets, gaseous ions, molecules ofthe nebulizing gas and of the atmosphere in the vicinity. Thepneumatically assisted spray is directed onto the surface of a samplematerial where it interacts with one or more analytes, if present in thesample, and generates desorbed ions of the analyte or analytes. Thedesorbed ions can be directed to a mass analyzer for mass analysis, toan IMS device for separation by size and measurement of resultingvoltage variations, to a flame spectrometer for spectral analysis, orthe like.

FIG. 11 illustrates schematically one embodiment of a DESI system 10. Inthis system, a spray 11 is generated by a conventional electrospraydevice 12. The device 12 includes a spray capillary 13 through which theliquid solvent 14 is fed. A surrounding nebulizer capillary 15 forms anannular space through which a nebulizing gas such as nitrogen (N₂) isfed at high velocity. In one example, the liquid was a water/methanolmixture and the gas was nitrogen. A high voltage is applied to theliquid solvent by a power supply 17 via a metal connecting element. Theresult of the fast flowing nebulizing gas interacting with the liquidleaving the capillary 13 is to form the DESI-active spray 11 comprisingliquid droplets. DESI-active spray 11 also may include neutralatmospheric molecules, nebulizing gas, and gaseous ions. Although anelectrospray device 12 has been described, any device capable ofgenerating a stream of liquid droplets carried by a nebulizing gas jetmay be used to form the DESI-active spray 11.

The spray 11 is directed onto the sample material 21 which in thisexample is supported on a surface 22. The desorbed ions 25 leaving thesample are collected and introduced into the atmospheric inlet orinterface 23 of a mass spectrometer for analysis by an ion transfer line24 which is positioned in sufficiently close proximity to the sample tocollect the desorbed ions. Surface 22 may be a moveable platform or maybe mounted on a moveable platform that can be moved in the x, y or zdirections by well known drive means to desorb and ionize sample 21 atdifferent areas, sometimes to create a map or image of the distributionof constituents of a sample. Electric potential and temperature of theplatform may also be controlled by known means. Any atmosphericinterface that is normally found in mass spectrometers will be suitablefor use in the invention. Good results have been obtained using atypical heated capillary atmospheric interface. Good results also havebeen obtained using an atmospheric interface that samples via anextended flexible ion transfer line made either of metal or aninsulator.

Low Temperature Plasma

Low temperature plasma (LTP) probes are described in Ouyang et al. (U.S.patent application Ser. No. 12/863,801 and PCT application numberPCT/US09/33760), the content of each of which is incorporated byreference herein in its entirety. Unlike electrospray or laser basedambient ionization sources, plasma sources do not require anelectrospray solvent, auxiliary gases, and lasers. LTP can becharacterized as a non-equilibrium plasma having high energy electrons,with relatively low kinetic energy but reactive ions and neutrals; theresult is a low temperature ambient plasma that can be used to desorband ionize analytes from surfaces and produce molecular ions or fragmentions of the analytes. A distinguishing characteristic of the LTP, incomparison with high temperature (equilibrium) plasmas, is that the LTPdoes not breakdown the molecules into atoms or small molecularfragments, so the molecular information is retained in the ionsproduced. LTP ionization sources have the potential to be small in size,consume low power and gas (or to use only ambient air) and theseadvantages can lead to reduced operating costs. In addition to costsavings, LTP based ionization methods have the potential to be utilizedwith portable mass spectrometers for real-time analytical analysis inthe field (Gao, L.; Song, Q.; Patterson, G. E.; Cooks, D. Ouyang, Z.,Anal. Chem. 2006, 78, 5994-6002; Mulligan, C. C.; Talaty, N.; Cooks, R.G., Chemical Communications 2006, 1709-1711; and Mulligan, C. C.;Justes, D. R.; Noll, R. J.; Sanders, N. L.; Laughlin, B. C.; Cooks, R.G., The Analyst 2006, 131, 556-567).

An exemplary LTP probe is shown in FIG. 12. Such a probe may include ahousing having a discharge gas inlet port, a probe tip, two electrodes,and a dielectric barrier, in which the two electrodes are separated bythe dielectric barrier, and in which application of voltage from a powersupply generates an electric field and a low temperature plasma, inwhich the electric field, or gas flow, or both, propel the lowtemperature plasma out of the probe tip. The ionization source of theprobe described herein is based upon a dielectric barrier discharge(DBD; Kogelschatz, U., Plasma Chemistry and Plasma Processing 2003, 23,1-46). Dielectric barrier discharge is achieved by applying a highvoltage signal, for example an alternating current, between twoelectrodes separated by a dielectric barrier. A non-thermal, low power,plasma is created between the two electrodes, with the dielectriclimiting the displacement current. This plasma contains reactive ions,electrons, radicals, excited neutrals, and metastable species in theambient environment of the sample which can be used to desorb/ionizemolecules from a solid sample surface as well as ionizing liquids andgases. The plasma can be extracted from the discharge region anddirected toward the sample surface with the force by electric field, orthe combined force of the electric field and gas flow.

In certain embodiments, the probe further includes a power supply. Thepower supply can provide direct current or alternating current. Incertain embodiments, the power supply provides an alternating current.In certain embodiments, a discharge gas is supplied to the probe throughthe discharge gas inlet port, and the electric field and/or thedischarge gas propel the low temperature plasma out of the probe tip.The discharge gas can be any gas. Exemplary discharge gases includehelium, compressed or ambient air, nitrogen, and argon. In certainembodiments, the dielectric barrier is composed of an electricallyinsulating material. Exemplary electrically insulating materials includeglass, quartz, ceramics and polymers. In other embodiments, thedielectric barrier is a glass tube that is open at each end. In otherembodiments, varying the electric field adjusts the energy andfragmentation degree of ions generated from the analytes in a sample.

Ionization Using Wetted Porous Material

Probes comprised of porous material that is wetted to produce ions aredescribed in Ouyang et al. (U.S. patent application Ser. No. 13/265,110and PCT application number PCT/US10/32881), the content of each of whichis incorporated by reference herein in its entirety. Exemplary probesare shown in FIGS. 13A-B. Porous materials, such as paper (e.g. filterpaper or chromatographic paper) or other similar materials are used tohold and transfer liquids and solids, and ions are generated directlyfrom the edges of the material when a high electric voltage is appliedto the material. The porous material is kept discrete (i.e., separate ordisconnected) from a flow of solvent, such as a continuous flow ofsolvent. Instead, sample is either spotted onto the porous material orswabbed onto it from a surface including the sample. The spotted orswabbed sample is then connected to a high voltage source to produceions of the sample which are subsequently mass analyzed. The sample istransported through the porous material without the need of a separatesolvent flow. Pneumatic assistance is not required to transport theanalyte; rather, a voltage is simply applied to the porous material thatis held in front of a mass spectrometer.

In certain embodiments, the porous material is any cellulose-basedmaterial. In other embodiments, the porous material is a non-metallicporous material, such as cotton, linen wool, synthetic textiles, orplant tissue. In still other embodiments, the porous material is paper.Advantages of paper include: cost (paper is inexpensive); it is fullycommercialized and its physical and chemical properties can be adjusted;it can filter particulates (cells and dusts) from liquid samples; it iseasily shaped (e.g., easy to cut, tear, or fold); liquids flow in itunder capillary action (e.g., without external pumping and/or a powersupply); and it is disposable.

In certain embodiments, the porous material is integrated with a solidtip having a macroscopic angle that is optimized for spray. In theseembodiments, the porous material is used for filtration,pre-concentration, and wicking of the solvent containing the analytesfor spray at the solid type.

In particular embodiments, the porous material is filter paper.Exemplary filter papers include cellulose filter paper, ashless filterpaper, nitrocellulose paper, glass microfiber filter paper, andpolyethylene paper. Filter paper having any pore size may be used.Exemplary pore sizes include Grade 1 (11 μm), Grade 2 (8 μm), Grade 595(4-7 μm), and Grade 6 (3 μm). Pore size will not only influence thetransport of liquid inside the spray materials, but could also affectthe formation of the Taylor cone at the tip. The optimum pore size willgenerate a stable Taylor cone and reduce liquid evaporation. The poresize of the filter paper is also an important parameter in filtration,i.e., the paper acts as an online pretreatment device. Commerciallyavailable ultra filtration membranes of regenerated cellulose, with poresizes in the low nm range, are designed to retain particles as small as1000 Da. Ultra filtration membranes can be commercially obtained withmolecular weight cutoffs ranging from 1000 Da to 100,000 Da.

Probes of the invention work well for the generation of micron scaledroplets simply based on using the high electric field generated at anedge of the porous material. In particular embodiments, the porousmaterial is shaped to have a macroscopically sharp point, such as apoint of a triangle, for ion generation. Probes of the invention mayhave different tip widths. In certain embodiments, the probe tip widthis at least about 5 μm or wider, at least about 10 μm or wider, at leastabout 50 μm or wider, at least about 150 μm or wider, at least about 250μm or wider, at least about 350 μm or wider, at least about 400μ orwider, at least about 450 μm or wider, etc. In particular embodiments,the tip width is at least 350 μm or wider. In other embodiments, theprobe tip width is about 400 μm. In other embodiments, probes of theinvention have a three dimensional shape, such as a conical shape.

As mentioned above, no pneumatic assistance is required to transport thedroplets. Ambient ionization of analytes is realized on the basis ofthese charged droplets, offering a simple and convenient approach formass analysis of solution-phase samples. Sample solution is directlyapplied on the porous material held in front of an inlet of a massspectrometer without any pretreatment. Then the ambient ionization isperformed by applying a high potential on the wetted porous material. Incertain embodiments, the porous material is paper, which is a type ofporous material that contains numerical pores and microchannels forliquid transport. The pores and microchannels also allow the paper toact as a filter device, which is beneficial for analyzing physicallydirty or contaminated samples. In other embodiments, the porous materialis treated to produce microchannels in the porous material or to enhancethe properties of the material for use as a probe of the invention. Forexample, paper may undergo a patterned silanization process to producemicrochannels or structures on the paper. Such processes involve, forexample, exposing the surface of the paper totridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane to result insilanization of the paper.

In other embodiments, a soft lithography process is used to producemicrochannels in the porous material or to enhance the properties of thematerial for use as a probe of the invention. In other embodiments,hydrophobic trapping regions are created in the paper to pre-concentrateless hydrophilic compounds. Hydrophobic regions may be patterned ontopaper by using photolithography, printing methods or plasma treatment todefine hydrophilic channels with lateral features of 200˜1000 μm. SeeMartinez et al. (Angew. Chem. Int. Ed. 2007, 46, 1318-1320); Martinez etal. (Proc. Natl Acad. Sci. USA 2008, 105, 19606-19611); Abe et al.(Anal. Chem. 2008, 80, 6928-6934); Bruzewicz et al. (Anal. Chem. 2008,80, 3387-3392); Martinez et al. (Lab Chip 2008, 8, 2146-2150); and Li etal. (Anal. Chem. 2008, 80, 9131-9134), the content of each of which isincorporated by reference herein in its entirety. Liquid samples loadedonto such a paper-based device can travel along the hydrophilic channelsdriven by capillary action.

Nano ESI (nESI)

Inductive nESI can be implemented for various kinds of nESI arrays dueto the lack of physical contact. Examples of circular and linear modesare illustrated in FIG. 13. In the rotating array, an electrode placed˜2 mm from each of the spray emitters in turn was supplied with a 2-4 kVpositive pulse (10-3000 Hz) giving a sequence of ion signals.Simultaneous ions signals were generated in the linear array usingpulsed voltages generated inductively in the adjacent nESI emitters.Nanoelectrospray spray plumes were observed and analytes are detected inthe mass spectrum, in both positive and negative detection modes.

INCORPORATION BY REFERENCE

Any and all references and citations to other documents, such aspatents, patent applications, patent publications, journals, books,papers, web contents, that have been made throughout this disclosure arehereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein.

EXAMPLES Example 1: Materials and Methods

Experiments were carried out using a custom built miniature massspectrometer (Mini 10; Gao et al., Anal. Chem. 2006, 78, 5994-6002) or aThermo LTQ mass spectrometer (Thermo Scientific, San Jose, Calif.).Capillary temperature: 150° C.; capillary voltage: 15 V; tube lensvoltage: 240 V. A custom power supply provided a pulsed output of50-5,000 Hz and 0-8 kV. DESI (Takats et al., Science 2004, 306, 471-473)conditions were: nitrogen gas 150 psi, a metal tube (id 250 μm, 5 cmlong) serves as outer electrode, an inner silica capillary serves as thespray emitter (id 50 μm), angle of DESI sprayer to substrate set at 40°,distance between spray tip and sample set at 2 mm, distance betweensample and MS inlet, 3 mm; the spray solution was MeOH/water (v:v=1:1),Commercial silica nanoelectrospray tips of 20 μm were obtained from NewObjective (Woburn, Mass., USA).

Example 2: On-Line Reaction Monitoring

Reaction monitoring using MS is of interest because of its chemicalspecificity, sensitivity and speed. There are many available ambientionization methods and there have been various attempts to use them foron-line reaction monitoring. Those systems exhibit weaknesses, such asonly being capable of off-line monitoring, which becomes ineffectivewhen fast reaction kinetics and short-lived reaction intermediates areinvolved, having practical drawbacks including severe capillary blockagewhen used for on-line monitoring of samples even with modestly highconcentrations of salts, and system set-ups that are all open-to-air,meaning that reactions sensitive to air/moisture are not currentlycontinuously monitored by MS.

This example shows an on-line reaction monitoring system using inductiveelectrospray ionization mass spectrometry (MS) to continuously monitorreacting chemical and biochemical systems to detect disappearance ofreagents and formation of products and to characterize the transientreaction intermediates. A specific embodiment used a sealed three-neckvessel in which the reaction solution was pressurized by inert gas toallow transfer continuously through a capillary to an emitter-spray tip.Ionization is produced by inductive electrospray ionization, asdescribed throughout. A positive electrospray potential applied to anelectrode near to but not in physical contact with the solution beingsprayed or the emitter is pulsed repeatedly to produce strong electricfields of both polarities which result in bursts of charged dropletsbeing emitted from the solution while avoiding direct physical contactbetween the high voltage and the reaction solution. Sheath gas was usedto help in the nebulization process and minimize size-variation in thedroplets. Sample splitting was useful in accelerating the flow rate ofsampling and decreasing the delay time while avoiding contamination ofthe MS inlet. Mass spectra recorded as a function of time gave thedesired kinetic information.

The inductive ESI-MS based monitoring system was used in real-timereaction monitoring, which is critical in process control to assurequality and purity in the manufacture of chemicals materials andpharmaceuticals. Monitoring precursors, intermediates and productsduring the reaction allows early detection of problems and overallproduct quality and facilitates yield improvement. Equally important isthe fact that the detailed information acquiring during on-line reactionmonitoring can help in optimizing reaction conditions and in elucidatingreaction mechanisms.

The inductive version of electrospray ionization mass spectrometry(inductive ESI-MS) was explored as a fast and simple way to monitorchemical reactions occurring in complex mixtures in real time. Thereactions chosen for study were the reductive amination of4-pyridine-carboxaldehyde with 4-(aminomethyl)pyridine, and Pd—Ccatalyzed hydrogenolysis of 3,4-dimethoxy-benzaldehyde. In inductiveESI, a positive electrospray potential is pulsed repeatedly to producestrong electric fields of both polarities which result in bursts ofcharged droplets being emitted from the solution while avoiding directphysical contact between the high voltage and the reaction solution. Thedelay time between sampling and analysis was reduced to several secondsusing a sample splitter while avoiding contamination of the MS inlet byspraying too large a volume of concentrated reaction solution. Kineticdata obtained by continuous inductive ESI-MS measurements of thereductive amination reaction were verified by intermittent proton NMR.Three short-lived intermediates were observed in the hydrogenolysisreaction, revealing a new triple intermediates pathway for thehydrogenolysis in methanol.

Introduction

Reaction monitoring is critical in process control to assure quality andpurity in the manufacture of chemicals and pharmaceuticals. Monitoringprecursors, intermediates and products during the reaction allows earlydetection of problems and overall product quality and yieldsimprovement. Equally important is the fact that the detailed informationacquiring during on-line reaction monitoring can help in optimizingreaction conditions and in elucidating reaction mechanisms. Modernmethods follow reaction progress by observing spectroscopic andelectrochemical methods, even though they have limited capabilities forstructure elucidation of products and intermediates present in complexmixtures.

Mass spectrometry (MS) coupled with various ionization methods is ofcurrent interest in monitoring reaction because of its structurecharacterization capability, high specificity and speed. Reactionmonitoring using MS involves sampling from the reaction system, ionizingand analyzing the mixture. Based on whether intermittent or continuoussampling is used, monitoring can be divided into off-line and on-lineversions with the latter being far more useful and more demanding.Off-line MS reaction monitoring has been done using a variety ofionization methods, electrospray ionization (ESI), matrix-assisted laserdesorption/ionization (MALDI), and direct analysis in real time (DART).This approach is useful for kinetic studies of slow reactions as well asproviding information on long-lived reaction intermediates. However, itbecomes ineffective when fast reaction kinetics and short-lived reactionintermediates are involved. By contrast, on-line MS monitoring doesapply in these circumstances but is much more difficult to implement. Analiquot of reaction solution must be continuously transferred from thereaction vessel to the MS for real-time analysis. Manual transfer ofsamples from the reaction system, as commonly done in off-line reactionmonitoring, is completely eliminated. As a result, on-line MS monitoringmust allow acquisition of real-time information on the reaction systemand intercept and characterize short-lived intermediates. Various ionsources have been investigated for this purpose including ESI,electrospray-assisted laser desorption/ionization (ELDI),ultrasound-assisted spray ionization (USI), extractive electrosprayionization (EESI), and low temperature plasma (LTP) ionization. However,severe limitations have been identified in every case.High-salt-containing sample solutions cannot be handled in conventionalESI sources, due to the likelihood of clogging of the ESI capillary.Heated columns used to vaporize the solution in EESI might expedite thereaction or lead to side products due to the higher temperature. Theopen-to-air setup of USI and LTP results in evaporation of reactionsolvent to the air and completely eliminates applications toair-sensitive or water-sensitive reactions. Substantial previous effortsin this lab using DESI and ESI-based methods for reaction monitoringhave had limited success for these reasons.

In inductive electrospray ionization, a potential is applied to a singleelectrode placed close to the capillary containing the reactionsolution. It pulses repeatedly in either the positive or negative modeat a frequency ranging from 10-2000 Hz. Strong dynamic electromagneticfields are produced in the ESI emitter resulting in a burst of chargeddroplets. The inductive ESI method is a variant of ESI but providesseveral new capabilities, such as being characterized by a remarkabletolerance to matrix effects and has a high efficiency. In conventionalESI source, clogging of the ESI capillary occurs quickly when ahigh-salt-containing sample is sprayed. By contrast, inductive ESI showsa remarkable resistance to deleterious effects of high saltconcentrations so much so that even urine and serum samples can beanalyzed directly. Inductive ESI uses a mono-polar applied potential andshould also not be confused with alternating current (AC) electrospray,which uses an alternating and directly applied potential.

AC electrospray has definite advantages but they do not extend to theremote application of the potential, the control of droplet creation orthe immunity to salts and complex matrices seen in inductive ESI.Because inductive ESI avoids physical contact between the electrode andthe spray solvent, there is no interference with reaction monitoring.Here, we describe the application of inductive ESI-MS to on-linereaction monitoring. The reaction solution was transferred to theemitter-spray tip by a capillary under positive helium pressure (orother gas) and ionized by applying inductively a pulsed DC voltage.Sheath gas was used to aid in the nebulization process and to minimizevariation in the droplet size. To allow rapid monitoring the delay timewas reduced to a few seconds by adding a sample splitter, whichexpedited sample transfer and prevented solution contamination of the MSinlet. Two reactions important in drug synthesis were selected formonitoring: (1) reductive amination of 4-pyridine-carboxaldehyde with4-(aminomethyl)pyridine (Scheme 1a), and (2) Pd—C catalyzedhydrogenolysis of 3,4-dimethoxy-benzaldehyde (Scheme 1b).

Apparatus for Inductive ESI-Based Reaction Monitoring

The configuration of the reaction monitoring system coupled with theinductive ESI ionization source is shown in FIG. 15. In a sealedthree-neck reaction vessel, one end of a silica capillary (i.d. 100 μm,length 40 cm) was immersed into the reaction solution. Reaction solutionwas pressurized by helium (˜5 psi) to allow transfer continuouslythrough the capillary to the emitter-spray tip. A gas bubbler filledwith a small amount of paraffin oil was used to monitor the reactionvessel pressure and exclude atmospheric oxygen and water from thereaction. A tee was added at the very front of the capillary tip toallow sheath gas to aid in the nebulization process and minimizesize-variation in the droplets. Without the sheath gas, the signal wasas much as 100 times poorer and more erratic. A three-way samplesplitter was added right after the sheath gas tee. An 8 cm long fusedsilica capillary, i.d. 100 μm, was connected through the sheath gas tee.The other outlet of the splitter was linked to a fused silica capillary,i.d. 200 μm, length 8 cm. A home-built power supply provided a positivepulsed output of 2000 Hz on the sheath gas. Strong electric fields wereproduced in the solution inside the emitter to give a burst of droplets.This procedure resulted in the pulsed emission of charged analytes fromthe reaction solution at a controlled pulse rate. The spray was directedtowards the inlet of the MS which was ca. 5 mm from the capillary tip.

Mass Spectrometry

A linear ion trap mass spectrometer (LTQ, Thermo Fisher Scientific, SanJose, Calif., USA) was used to record positive ion mode mass spectra.Typical MS parameters used included averaging of 3 microscans, 100 msmaximum injection time, 15 V capillary voltages, 150° C. capillarytemperature, and 65 V tube lens voltage. Data were acquired andprocessed using Xcalibur 2.0 software (Thermo Fisher Scientific, SanJose, Calif., USA). The identification of analyte ions was confirmed bytandem mass spectrometry (MS/MS) using collision-induced dissociation(CID). An isolation window of 1.5 Th (mass/charge units) and normalizedcollision energy of 30%-40% (manufacturer's unit) was selected for theCID experiments.

¹H NMR Spectroscopy

¹H NMR spectra were acquired using a Varian Inova-300 spectrometer(Department of Chemistry, Purdue University). Chemical shifts arereported in parts per million (ppm) using CD3OD as the reference peak.

Chemicals and Reagents

All reagents and solvents used were of analytical grade or higher andwere used directly without further purification.4-(Aminomethyl)pyridine, 4-pyridine-carboxaldehyde, sodium borohydride,3,4-dimethoxy-benzaldehyde, palladium (5 wt. % on activated carbon) andHPLC grade methanol (MeOH) were purchased from Sigma-Aldrich (St. Louis,Mo., USA). Water was purified and deionized by a Milli-Q system(Millipore, Bedford, Mass., USA).

Synthesis of bis((pyridin-4-yl)methyl)amine (4) and Reaction Monitoring

To a three-neck round-bottom flask under He atm was added a solution of1 (507 μL, 5 mmol) in 50 mL of MeOH, and then 2 (517 μL, 5.5 mmol) wasinjected. After stirring for 46 min, a solution of NaBH₄ (95 mg, 2.5mmol) in 2 mL of H2O was injected to the reaction mixture. The resultingreaction mixture was stirred for 50 min. The whole reaction process wasmonitored by inductive ESI-MS which served to characterize intermediatesand products.

Measurement of Delay Time

To a 50 mL of MeOH was injected 1 (507 μL, 5 mmol). The interval betweeninjection of 1 into the solution and the time when the peak at m/z 109was seen was recorded as the delay time. The whole process was monitoredby inductive ESI-MS with capillary of different i.d. size and with orwithout a splitter.

Synthesis of 1,2-dimethoxy-4-methylbenzene (6) and Reaction Monitoring

The three-neck reaction flask used to monitor formation of 4 wasequipped with a syringe filter on a syringe tube in the middle neck inorder to prevent the Pd—C particles from blocking the capillary. Thecapillary for continuous sampling was inserted into the syringe tube andsucking the solution filtered. To a 46 mL of MeOH in the vessel wasadded 5% Pd—C (1.6 mg, 6 wt %). The air in the vessel was replaced byH₂. Instead of using a He cylinder, H₂ cylinder was used to provide H₂continuously for hydrogenolysis. The precursor (5) (26 mg, 0.16 mmol) in3 mL of MeOH was injected into the solution followed by adding aq HCl (1μL) in 1 mL of MeOH. After 80 min, twice the amount of the Pd—C inmethanol was added in small aliquots to the reaction mixture. Theresulting reaction mixture was stirred for another 70 min. The wholereaction process was monitored by inductive ESI-MS.

On-Line Monitoring of Reductive Amination of 4-pyridine-carboxaldehydewith 4-(aminomethyl)pyridine

In this Example, reductive amination of 4-pyridine-carboxaldehyde (2)with 4-(aminomethyl)pyridine (1) was carried out using a one-pottwo-step protocol to produce bis((pyridin-4-yl)methyl)amine (4).Time-resolved mass spectra of the aldehyde-amine condensation step andthe imine reduction step are shown in FIG. 16. Ions were identifiedaccording to their m/z values and confirmed by tandem mass experiment.Reactant 1 was first injected into the methanol reaction solvent. Ionsof protonated 1 at m/z 109 were detected at 0.4 min later, on the heelsof which the protonated dimer at m/z 217 appeared and reached its steadystate within 4.8 s. (FIG. 16 panels A-B) After reactant 2 was injectedinto the reaction solution and the protonated methanol adduct ions ofthe monomer and dimer of 2 at m/z 140 and 279 showed up. The ions of theintermediate (pyridin-4-yl)-N-((pyridin-4-yl)methylene)methanamine (3)at m/z 198 and 230 formed quickly and the intensity increased sharply(FIG. 16 panel C). The intensity of ions of 1 decreased but the ions didnot disappear, which corresponded to the fact that this is anequilibrium reaction (FIG. 16 panel D). About 50 min later, sodiumborohydride solution was injected into the system. The sodiated adductions of product 4 at m/z 222 started to appear, while the intermediateions of 3 decreased a lot (FIG. 16 panel E). Ten minutes later, the peakof the product 4 was reaching its highest intensity (FIG. 16 panel F).The selected ion chronogram shows dynamic changing of ions correspondingto the precursor 1, intermediate 3 and product 4 (FIG. 17). The kineticprocess of reductive amination can be clearly seen. The firstcondensation step of this reaction happened very fast; once 2 was addedto the solution of 1, it took less than 1 min for the reaction to reachequilibrium. The intermediate 3 was generated simultaneously with thedecrease of 1. The second reduction step was not as fast as the firststep. With the reduction of intermediate 3, the first step was driven tothe right. Thus ions of 1 were depleted. Altogether, it took around 10min for the product 4 to reach its highest intensity. The totalmonitoring time was 90 min without any clogging in the fused-silicacapillary using high concentrations of reaction solution that arerepresentative of those of industrial interest.

The above kinetic data obtained by inductive ESI-MS monitoring werecompared to that collected through ¹H NMR monitoring to verify theaccuracy. Reductive amination of 2 with 1 was studied under the sameconditions but monitored by ¹H NMR. Because the reaction cannot bemonitored continuously, we chose several time points and sampledaliquots of the solution for NMR analysis. Hydrogens from the methylenegroups in compounds 1, 3, 4 have different chemical shifts (4.8 ppm, 5.5ppm and 4.6 ppm, respectively) due to the differences in the adjacentN-containing functional groups. Therefore the hydrogen signals of themethylene groups were chosen as the characteristic marker for eachcompound. The product yield at each selected time point was calculatedas the ratio of methylene peak area for compound 4 vs the total for 1, 3and 4. The yield is shown as a kinetic curve in FIG. 18. Comparing thekinetic curves obtained by inductive ESI-MS and ¹H NMR, the same timetrends are shown. The kinetic data acquired by inductive ESI-MS providedcontinuous and more detailed information of the reaction. The reactionselected here was relatively simple; in other reactions there might bemore than four main compounds. This would represent a challenge for ¹HNMR owing to the difficult peak assignments in mixture, while even inthat case each selected ion chronogram could be still obtained from MS.

The time lag between sampling and measuring compounds in a reactionmixture, is an important factor that should be taken into account inreaction monitoring, because it affects accuracy of kinetic data anddetermines how quickly the operator can respond to an unexpectedsituation. In off-line MS reaction monitoring, delay time emerging fromsampling, sample preparation and analysis process is various,uncontrollable and always lengthy. In on-line MS reaction monitoring,the sample is transferred directly from the reaction system to the MS.The MS result thus represents the state of the reaction systemcontinuously with a short offset for solution transport which is oftendone by a capillary. Under certain vessel pressure, the length and theinner diameter of the chosen capillary determines the duration time ofthe delay, which reduces uncertainty of measuring the delay time.

In this Example, delay time of inductive ESI-MS monitoring system wasmeasured using capillaries of various inner diameters but the samelength (40 cm). The selected ion chronogram of ions of 1 illustrates thedelay time after reagent addition (FIG. 19). The delay time for i.d. 50μm is 1.2 min and this was decreased to 2.4 s when using a capillary ofi.d. 180 μm. In this case, however, the consumption of the reactionsolution for electrospray was as high as 0.19 mL/min. The large sprayvolume of analytes could contaminate the MS inlet. In order to decreasethe amount of analytes into MS while maintain the short delay time, athree-way sample splitter was added before the nebulizing gas tee. Acapillary with double the inner diameter (i.d. 200 μm) was used for thesplitter outlet that accelerated the replacement of solution inside thetransferring capillary. The arrival time in the splitter equipped systemwas 0.2 min faster than that without a splitter tee. Thus, the flow rateof sampling was increased, while the volume of solution used forionization in the emitter was still kept the same as that delivered by a100 μm capillary which effectively avoided contamination of MS inlet.

On-Line Monitoring of Pd—C Catalyzed Hydrogenolysis of3,4-dimethoxy-benzaldehyde

Pd—C catalyzed hydrogenation has been widely employed in numerousorganic transformations (Merz et al., M. Synthesis 1993, 797-802; andSagar et al., Bioorg. Med. Chem. 2004, 12, 4045-4054). The reactioncommonly proceeds under H₂ atmosphere, which requires the reactionsystem pressure-tight or within a positive pressure of H₂. So far,almost no available MS monitoring system is possible for monitoring thistype of reaction. Inductive ESI-MS reaction monitoring is using a sealedflask with positive pressure inside, which is able to expand the scopeto monitoring reaction involving gas as a reactant or reactantssensitive to air and water. Here, Pd—C catalyzed hydrogenolysis of3,4-dimethoxybenzaldehyde was chosen to be monitored by inductiveESI-MS.

Time-resolved mass spectra of Pd—C catalyzed hydrogenolysis of3,4-dimethoxybenzaldehyde are shown in FIG. 20. Ions were determinedbased on their m/z values and tandem MS. At 0.48 min after precursor 5was injected into the Pd/C suspension of methanol, its ions [M₅+H]⁺ atm/z 167 and [M₅+Na]⁺ at m/z 189 were detected and the reaction started(FIG. 20, panel A). Within 0.02 min, a new peak at m/z 191 started toappear. It reached its highest intensity within 0.4 min and disappeared0.15 min later (FIG. 20, panel B). Another two new peaks at m/z 235 and181 was shown 0.34 min and 0.38 min behind the peak at m/z 191respectively (FIG. 20, panel C-D). 0.3 min later, the peak at m/z 235disappeared while the peak at m/z 181 dominated the reaction (FIG. 20,panel E-F). At the same time, the ion of hydrogenolysis product 6 at m/z151 began to form. It became base peak at 18 min, and dominated thereaction at 45 min (FIG. 20, panel G). The whole reaction process showedthere were three intermediates (m/z 191, 235 and 181) produced beforethe formation of the hydrogenolysis product.

The trap of transient intermediates in the Example of reaction mechanismis significantly important, while their short lives challenge theclassic way of monitoring reaction such as TLC, HPLC and GC whichrequires a certain time for analysis. Online inductive ESI-MS monitorsthe reaction continuously and provides instant structure information forall of the analytes in the mixture solution, therefore it has thecapability to catch the transient intermediates. The kinetics of Pd—Ccatalyzed hydrogenolysis of benzaldehydes in methanol was provided inFIGS. 21A-B, showing changing curves of ions corresponding to theprecursor 5, three intermediates 7, 8, 9, and the product 6. Fromselected ion chronogram at time range of 2.5-3.5 min (FIG. 21B), theintermediate ion at m/z 191 was formed firstly among the threeintermediates and almost at the same time when the precursor 5 wasadded. The appearance of its peak centralized in 2.9-3.05 min. Theintermediate ion at m/z 235 was shown followed by m/z 191 and appeared93% of its peak area in 2.9-3.2 min. The life time of these twointermediates was as short as less than 0.3 min. With the decrease ofthe former intermediates, the intermediate ion at m/z 181 increased, bythe conversion of which the product 6 was finally formed.

Pd—C catalyzed hydrogenolysis of benzaldehydes to methylbenzenes hasbeen described as proceeding via benzenemethanol intermediate pathway(Nishimura et al., S. Handbook of heterogeneous Catalytic Hydrogenationfor Organic Synthesis; John Wiley & Sons: New York, N.Y., 2001; Chapter5; Connolly et al., J. Med. Chem. 1996, 39, 46-55) becausebenzenemethanol was often isolated as an intermediate or as a majorby-product. In the above case, intermediate ions at m/z 191, 235 and 181were assigned as sodiated adduct ions of dimethoxy benzenemethanol (7),sodiated adduct ions of dimethoxy benzaldehyde dimethyl acetal (9) andhydride abstraction ions of dimethoxy benzyl methyl ether (8),respectively. In agreement with the classic benzenemethanol pathway,3,4-Dimethoxybenzaldehyde was firstly hydrogenated to dimethoxybenzenemethanol (m/z 191), then converted to dimethoxy benzyl methylether (m/z 181), and finally dimethoxy toluene (m/z 151) was produced.However, this could not explain the appearance of dimethoxy benzaldehydedimethyl acetal (m/z 235). The observed sodiated adduct ions dimethoxybenzaldehyde dimethyl acetal suggested a second new pathway ofhydrogenolysis where benzaldehyde acetal was produced as an intermediateinstead of benzenemethanol. Recently, the similar benzylaldehyde acetalintermediate was separated and confirmed (Merz et al., Synthesis 1993,797-802). Although, that work claimed that when lower alcohols (methanolor ethanol) were used as solvents, only benzylaldehyde acetalintermediate rather than benzenemethanol was produced as the firstintermediate. Instead, a new two-way three-stage pathway involving threeintermediates 7, 8, 9 was proposed here as shown in scheme 2, by whichthe functions of each reaction intermediate were well recognized.

The observed cations at m/z 181 and 151 were one mass unit lower thanthe molecular weight of compound 8 and 6 respectively, and so correspondto loss of hydride from the neutral molecules. The key step in ESI isnormally the generation of the protonated form of the molecule:M+AH⁺→[M+H]⁺+A. However, hydride abstraction is well known route topositive ion formation in chemical ionization mass spectrometry. It ispossible the presence of trace Pd/C particles affect the outcome of theencounter between a proton donor and a molecule in the present ESIexperiments favoring hydride loss by providing a route to H₂ take up asdiscussed later.

When Pd—C catalyzed hydrogenolysis of 3,4-dimethoxybenzaldehyde inmethanol was studied for the first time, the reaction was stopped whenions at m/z 151 dominated the reaction (FIG. 20 panel G). However, whenthe reaction mixture was kept overnight and re-examined by inductiveESI-MS again the next morning, the peak at m/z 151 had almostdisappeared, being replaced by a peak at m/z 153. In order to determinewhether they were the different compounds or the same ones subjected todifferent ionization methods, two reaction mixtures were prepared for ¹HNMR, one dominated by the ions at m/z 151 and the other dominated by theions at m/z 153, respectively. The ¹H NMR results confirmed theformation of the same product 6. Therefore m/z 151 was assigned to[M−H]⁺, while m/z 153 represents [M+H]⁺. In order to explore therelationship of these two ionized forms of what is apparently the sameneutral product, the kinetics was monitored continuously (FIG. 20 panelA). After 80 min, twice the initial amount of Pd/C in methanol was addedto the reaction mixture. At 120 min, the ions at m/z 153 finally becamethe dominant peak (FIG. 20 panel H). During the long time interval whenthe peak at m/z 151 was decreasing and that at m/z 153 was increasing,the two peaks displayed a one-to-one correspondence, which againsuggested they were generated from the same compound. We speculate thatas intermediate 8 was consumed and converted to product 6, thereremained no substrates for hydrogenation in the system. The surface ofthe Pd/C then became saturated with H₂ which eliminated the drivingforce of hydride abstraction. The formation of the protonated ratherthan the deprotonated form of the product occurred and dominated themixture in the end.

Conclusions

Inductive ESI-MS was a useful tool for direct and continuous monitoringof organic reactions in situ while avoiding any need for physicalcontact of the high voltage with the reaction solution. Sheath gas wasused to help in the nebulization process and minimize size-variation inthe droplets. Sample splitting was useful in accelerating flow rate ofsampling and decreasing the delay time while avoiding contamination ofthe MS inlet. The kinetic study of reductive amination monitored byinductive ESI-MS compared well with that acquired off-line by ¹H NMR,which verified that inductive ESI-MS reaction monitoring systemreflected the dynamic trend of the reaction. It also provided moredetailed information than ¹H NMR. Online monitoring Pd—C catalyzedhydrogenolysis of 3,4-dimethoxy benzaldehydes was successfully monitoredin the sealed vessel with positive H₂ supplying system. Hydrideabstraction ion [M−H]⁺ was observed in Pd/C involved reaction andpossible reasons were provided.

On-line monitoring of the reaction by inductive ESI-MS permitted themonitoring of how the reaction proceeded, and allowed for the detectionand characterization of the transient reaction intermediates, whichprovides a general and efficient way to investigate the reactionmechanism. The system is applicable to reactions that are sensitive toair or water. Moreover, since inductive ESI has been proved tofacilitate high throughput detection, the system has the possibility tomonitor several parallel reactions at the same time by multiplexingreactions and analyzing with a single mass spectrometer.

Example 3: Solvent Compatibility

In on-line reaction monitoring, the solvent used for conducting thereaction is directly flowed to an ion generating device for productionof the ions that are analyzed in real-time. Therefore, in on-linereaction monitoring, the reaction solvent is also the ionizationsolvent. Importantly, the solvent used in on-line reaction monitoringmust be compatible as both the reaction solvent and the ionizationsolvent for on-line reaction monitoring to work. Choosing a reactionsolvent that is not capable of also being an ionization solvent resultsin an inability to generate ions of reaction intermediates and finalproducts in real-time. On the other hand, choosing a reaction solventthat works well for ionization, but is not compatible with the reactionto be conducted results in an inability to conduct the reaction. Thisaspect of the invention is exemplified below.

The Negishi reaction, palladium-catalyzed cross-coupling oforganohalides with organozinc reagents, is a selective and versatilemethod for the formation of C—C bonds that has been widely used infields ranging from medicine to agriculture and materials development.The reagents involved in the Negishi reaction are extremely susceptibleto the presence of water or oxygen, as a result the reaction needs to beperformed with strict exclusion of water and air. This represents achallenge to any on-line monitoring system. Due to that difficulty,on-line reaction monitoring has not been used for any air and/or watersensitive reactions. The Negishi reaction is also of interest because ofa series of cascaded reaction intermediates, the instability andtransience of which prevents their isolation and purification. Massspectrometry is especially suitable for following the rapidtransformations of the intermediates without separation.

In this Example, cross coupling of 3-bromoquinoline with diethyl zinccatalyzed by Pd-PEPPSI (palladium-pyridine, enhanced precatalyst,preparation, stabilization and initiation) in tetrahydrofuran (Scheme 1)was monitored by inductive ESI-MS.

The kinetics of the Negishi cross coupling is shown in FIG. 22, in whichthe changing abundances of the total ion current, and the currentscorresponding to the 3-bromoquinoline (m/z 208), the Pd-PEPPSI product(m/z 389), a representative intermediate (m/z 407) and the reactionproduct ethylquinoline (m/z 158) are displayed. At 8.4 min after3-bromoquinoline was injected into a solution of catalyst Pd-PEPPSI, theprotonated ions of 3-bromoquinoline at m/z 208 and 210 were detected.With the injection of diethyl zinc reagent over the period 11.6-14 min,catalyst Pd-PEPPSI was activated from Pd (II) to Pd (0) and ions at m/z389 derived from the ligated N-heterocyclic carbine showed up at 12.2min. Protonated 3-bromoquinoline began to decrease at 12 min, whileseveral reaction intermediates were detected including major ions at m/z407, 379 and 250 (FIG. 23) before the reaction product ions due toprotonated ethylquinoline at m/z 158 gained their sharp increase. Theintermediates began to decrease at 15 min and at the same time thereaction product ethylquinoline increased steadily and reach its highestintensity at 22 min.

In the online monitoring system for this reaction, the reaction solventalso serves as the spray solvent in order to exclude air and moistureand avoid introduction of interference of unnecessary ions. When usingspray-based ionization methods, the chosen solvent has an effect on theionization performance of an analyte of interest, which may beattributed to the properties of the solvent including surface tension,solvent polarity and vapor pressure. In this Example, anhydroustetrahydrofuran (THF) was used as the reaction and spray solvent foronline reaction monitoring.

Without consideration for the reaction, methanol or methanol/watermixture would be best for ionization. However, those solvents would notbe compatible as reaction solvents. Using the same setup as in thisExample, methanol and methanol/water were used as solvents for onlinemonitoring of a Negishi reaction. It was found that the intensity,continuity and stability of recorded MS signal were much worse.

What is claimed is:
 1. An online reaction monitoring system, the systemcomprising: a reaction vessel comprising an outlet port; a channel thatextends from within the outlet port; an electrode operably associatedwith a distal end of the channel, wherein the electrode is external tothe channel and does not physically contact the channel or a liquid orvapor within the channel; and a mass spectrometer.
 2. The systemaccording to claim 1, wherein the electrode is positioned to inductivelyinteract with a distal end of the channel.
 3. The system according toclaim 1, wherein the reaction vessel comprises one or more inlet ports.4. The system according to claim 3, wherein the system further comprisesa gas source operably coupled to the reaction vessel through one of theone or more inlet ports.
 5. The system according to claim 1, wherein thechannel extends into the reaction vessel.
 6. The system according toclaim 1, wherein the channel comprises one or more splitters.
 7. Thesystem according to claim 6, wherein a first splitter withdraws aportion of a liquid from the channel.
 8. The system according to claim7, wherein a second splitter is coupled to a nebulizing gas source inorder to introduce a nebulizing gas into the channel.
 9. The systemaccording to claim 1, further comprising a heating element operablyassociated with the reaction vessel.
 10. The system according to claim1, wherein the mass spectrometer is a miniature mass spectrometer. 11.An online reaction monitoring system, the system comprising: a reactionvessel comprising an outlet port; a channel that extends from within theoutlet port; an electrode positioned to inductively interact with adistal end of the channel, wherein the electrode is external to thechannel and does not physically contact the channel or a liquid or vaporwithin the channel and the electrode produces a pulsed current; and amass spectrometer.
 12. The system according to claim 11, wherein thepulsed current is a pulsed DC current.
 13. The system according to claim11, wherein the reaction vessel comprises one or more inlet ports. 14.The system according to claim 13, wherein the system further comprises agas source operably coupled to the reaction vessel through one of theone or more inlet ports.
 15. The system according to claim 11, whereinthe channel extends into the reaction vessel.
 16. The system accordingto claim 11, wherein the channel comprises one or more splitters. 17.The system according to claim 16, wherein a first splitter withdraws aportion of a liquid from the channel.
 18. The system according to claim17, wherein a second splitter is coupled to a nebulizing gas source inorder to introduce a nebulizing gas into the channel.
 19. The systemaccording to claim 11, further comprising a heating element operablyassociated with the reaction vessel.
 20. The system according to claim11, wherein the mass spectrometer is a miniature mass spectrometer.